The objective of the proposed research is to elucidate the mechanisms of inactivation, degradation, and turnover of neuronal nitric oxide synthase (nNOS), a cytochrome P450-like hemoprotein, caused by exposure to xenobiotics, including drugs and environmental toxicants. The central hypothesis is that such effects diminish the ability to form NO, an important bioregulatory molecule involved in many physiological functions, and cause some forms of chemically induced toxicities. It was recently shown that guanabenz (WytensinTM), an antihypertensive agent, inactivates penile nNOS in vitro in a metabolism dependent manner and causes a loss of nNOS protein and activity in vivo. Similarly, cigarette smoke contains compounds that lead to metabolism-based inactivation and loss of penile nNOS in vivo. These findings may be important as NO plays a key role in penile erection and antihypertensive agents as well as cigarette smoking are known to cause impotence. Several metabolism-based in activators, including guanabenz, were found to cause the enhanced proteolytic degradation of nNOS that, in part, causes the loss of nNOS protein in vivo. This degradation was found to involve hsp9o-based chaperones, ubiquitination, and proteasomal degradation. Thus, the aims of the current proposal are to understand how nNOS is inactivated and covalently altered by xenobiotics and how the steady state levels of nNOS are subsequently affected. This will involve the study of not only the degradation process but also the opposing forces of repair or assembly of nNOS. We will utilize modern chemical and biological techniques in vitro, cellular, and in vivo models to address these aims. The specific aims are: (1) To determine the molecular mechanism of nNOS inactivation caused by metabolism-based in activators, (2) To characterize the structural determinants that render nNOS susceptible to ubiquitination and proteasomal degradation, (3) To characterize the processes responsible for activation or maintenance of nNOS function, (4) To characterize the effect of tobacco smoke constituents on nNOS with the use of knowledge gained in Aims 1-3. These studies should lead to a better understanding of how drugs inactivate and regulate nNOS and thereby lead to the design of safer drugs without undesired toxicological side effects, such as impotence. Conversely, the same knowledge could be used to design safer and more effective inhibitors of NOS for pharmacological use in a variety of neurological diseases.